Tryptophan Photolysis Leads to a UVB‐lnduced 66 kDa Photoproduct of Ribulose‐1,5 Bisphosphate Carboxylase/Oxygenase (Rubisco) In Vitro and In Vivo

Abstract

Abstract— Proteins are vulnerable to environmental UVB (290‐320 nm) because aromatic amino acids, particularly Trp, absorb in this spectral region. We have shown previously that UVB impacts on ribulose‐l,5‐bisphosphate carboxylase/oxygenase (Rubisco), resulting in the formation of a 66 kDa photoproduct in vivo (Wilson et al, Plant Physiol. 109,221–229, 1995). To determine if Trp photolysis is involved in the production of this specific protein photoproduct, the effects of UVB on a homogeneous preparation of Rubisco were examined. A UVB photoproduct of 66 kDa, identical to the in vivo product, was formed in vitro. The 66 kDa product was shown by immunological methods to be a cross‐link between a large subunit (53 kDa) and a small subunit (14 kDa). Time‐resolved Trp fluorescence was used to demonstrate that a Trp fluorescence signal is lost with kinetics that mirror the rate of formation of the 66 kDa photoproduct, indicating that a Trp photolysis step is involved in the mechanism of photoproduct formation. The relative rates of both Trp photolysis and 66 kDa photoproduct formation did not change with Rubisco concentration, consistent with a monomolecular reaction that would occur between sub‐units within a Rubisco holoenzyme complex. Finally, formation of the 66 kDa photoproduct was found to be pH dependent.